Caprine arthritis-encephalitis virus-infected goats can generate human immunodeficiency virus-gp120 cross-reactive antibodies1

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Virology 315 (2003) 217–223

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Caprine arthritis-encephalitis virus-infected goats can generate human immunodeficiency virus-gp120 cross-reactive antibodies1 Katherine A. Louie,a Joseph M. Dadgari,a Bethany M. DeBoer,a Hilary Weisbuch,a Peter M. Snow,b William P. Cheevers,c Angeline Douvas,a,1 and Minnie McMillana,* a

Departments of Microbiology, Neurology, and Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA b Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA c Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, USA Received 8 April 2003; returned to author for revision 21 May 2003; accepted 23 June 2003

Abstract Lentiviruses display surprisingly disparate clinical manifestations in their specific hosts, share complex genetic structures, and exhibit extensive diversity, particularly in their envelope genes. The envelope protein, gp135, of caprine arthritis-encephalitis virus (CAEV) has minimal primary sequence homology to gp120, the envelope protein of human immunodeficiency virus (HIV). Nevertheless, they bear certain similarities in that they both possess five variable regions, both are heavily glycosylated, and both share short sequence motifs. We establish a further relationship and demonstrate that some goats, infected with CAEV, possess gp135-specific antibodies which cross-react with gp120 from several HIV strains, provided the protein is expressed in insect cells. We show that, although the cross-reactivity of these immunoglobulins depends on the level of glycosylation, nevertheless, some antibodies recognize the protein epitopes on gp120, at least some of which are linear in character. Further characterization of this unexpected cross-reaction will define its potential therapeutic utility. © 2003 Elsevier Inc. All rights reserved. Keywords: Lentivirus; HIV; CAEV; Antibody cross-reactivity; gp135 envelope protein; gp120 envelope protein; Glycosylation

Introduction Lentiviruses exhibit dramatically disparate manifestations of disease in their specific hosts (Clements and Zink, 1996; Coffin, 1996). While human immunodeficiency virus (HIV) infects and cripples the human immune system with devastating consequences, caprine arthritis-encephalitis virus (CAEV) causes chronic inflammation, with infected goats often suffering from arthritis (Cheevers and McGuire, 1988; Crawford et al., 1980; Levy, 1998). Despite these obvious differences, lentiviruses share many biological properties, including similar structural and genetic organi-

* Corresponding author. USC/Norris Cancer Center, Room 6340, 1441 Eastlake Ave., Los Angeles, CA 90033. Fax: ⫹1-323-865-0099. E-mail address: [email protected] (M. McMillan). 1 We dedicate this article to the memory of Angeline Douvas. 0042-6822/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0042-6822(03)00523-3

zations and extensive genetic diversity (Clements and Zink, 1996; Miller et al., 2000). Viral entry into target cells is mediated by the envelope glycoprotein. Its precursor is proteolytically cleaved in the Golgi complex and the surface unit (SU) or envelope protein itself remains noncovalently associated with the transmembrane subunit on both the surface of infected cells and the virions. The envelope protein of HIV is gp120, and it has been the subject of intensive research (Poignard et al., 2001). It displays considerable conformational flexibility and is one of the most extensively glycosylated proteins known (⬃50% by weight) (Leonard et al., 1990). This diminishes its immunogenicity and effectively conceals receptor-binding sites (Poignard et al., 2001). Comparison of the primary sequences of gp120 variants reveals conserved domains interrupted by five regions of variability (V1–V5) (Modrow et al., 1987). V1–V4 are large surface-exposed

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loops, while the conserved regions form a core containing many elements important for receptor binding (Leonard et al., 1990; Poignard et al., 2001). During viral entry, gp120 binds to the CD4 receptor molecule. This contributes to attachment of the virus to the target cell and triggers a conformational change that allows high-affinity binding to the chemokine coreceptor. At present, the 3D structural information on gp120 is limited to this CD4-associated configuration of the core of gp120 (Kwong et al., 1998; Rizzuto et al., 1998; Wyatt et al., 1998; Wyatt and Sodroski, 1998). In contrast to gp120, no 3D structural information exists for gp135, the SU of CAEV, nor has its receptor been characterized. Although gp135 comprises an additional ⬃70 amino acids including four cysteine residues, nevertheless, it bears similarities to gp120 in that it, too, is heavily glycosylated and possesses five variable regions. In addition, gp135 and gp120 share short sequence motifs (Crawford et al., 1980; Hotzel and Cheevers, 2000, 2001; Knowles et al., 1991; Ozyoruk et al., 2001; Valas et al., 2000). In this article, we identify a further relationship by demonstrating that gp135-specific antibodies are capable of cross-reacting with gp120.

Results Sera from some goats infected with CAEV react with baculovirus-derived HIV-gp120 protein We used an ELISA assay to monitor the antibody reactivities of 46 goats living on ranches in southern and central California which were known to harbor CAEV. The goat sera were tested for reactivity to the envelope proteins gp135-63, derived from CAEV-63, the prototype isolate of the virus (Crawford et al., 1980), HIV gp120 IIIB protein isolated from insect cells (gp120 IIIB-r), and HIV gp120 IIIB protein isolated from mammalian cells (gp120 IIIB-m). The envelope protein from simian immunodeficiency virus (SIV) also produced in insect cells, and ovalbumin protein (OVA) were used as negative controls. Goat anti-gp120 IIIB serum and a mouse anti-gp120 monoclonal antibody were used as positive controls. In general, the goat sera fell into three categories: gp135-positive/gp120 IIIB-r-positive, gp135 positive/gp120 IIIB-r negative, or gp135 negative/ gp120 IIIB-r negative. These data are summarized in Fig. 1, where absorbance relating to gp120 is plotted on the abscissa and that relating to gp135-63 is on the ordinate. The background values for the SIV protein have been subtracted (⬍0.38 OD units at 405 nm). We found that of the 46 goats, 21 tested positive for gp135-63 and, of those, 8 were positive for gp120 IIIB-r protein. Interestingly, 24 goats, although raised in a CAEV-infected environment, were negative for gp135 and for gp120 IIIB-r, while only 1 goat of 46 was gp120-positive/gp135-negative. This goat was also

Fig. 1. A subset of CAEV-infected goats possess gp120-reactive antibodies. Results of indirect ELISA assays testing goat sera (1:50 dilution) against the following antigens: gp135-63, gp120 IIIB-r, and SIV envelope proteins. Background SIV absorbance was subtracted from both the gp135 and the gp120 values for each serum.

negative for CAEV gag-specific antibodies (data not shown). Since this goat cohabited with other goats which were unequivocally infected with CAEV, it, too, may have been infected and not have yet seroconverted. Alternatively, the reactivity may be due to other unknown factors. In Fig. 2 we present detailed ELISA results from representative goats within each category. Serum from goat 4g2 is positive for gp135-63 and for gp120 IIIB-r, but negative for gp120 IIIB-m and SIV. We also show the results from a CAEV-negative (2g1) and a CAEV-positive goat (19g1) which does not possess gp120-reactive antibodies (Figs. 2b and c). Both the goat anti-gp120 IIIB serum and the mouse anti-gp120 monoclonal antibody recognize gp120 IIIB-r, as well as gp120 IIIB-m (Figs. 2d and e). To determine whether the goat antibodies are specific only for the gp120 IIIB protein or can react with gp120 derived from other strains of HIV, we used gp120 MN-r, gp120 CM-r, and gp120 SF2-m as antigens in an ELISA assay. A typical result, using 4g2 serum, is shown in Fig. 2f. This serum reacts with both gp120 MN-r and gp120 CM-r proteins, but does not react with gp120 SF2-m isolated from mammalian cells. We conclude from the results of these ELISA assays that several CAEV-infected goats possess antibodies which interact with gp120 from several viral strains, provided the protein is expressed in insect, not mammalian, cells. Envelope-specific antibodies generated by the 4g2 goat cross-react with gp120 IIIB protein We next wished to establish whether the antibodies generated by the 4g2 goat to the gp135 envelope protein also interacted with the gp120 protein, i.e., they were crossreactive or, alternatively, comprised two separate populations of immunoglobulins.

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Fig. 2. A typical ELISA experiment demonstrating that the CAEV-infected goat, 4g2, possesses antibodies which interact with gp120 from several HIV strains. The symbols which signify antigens are shown in the right-hand panel, and the identity of the antibody used is shown in the top left-hand corner of each panel. Serum dilution is indicated on the abscissa and the absorbance at 405 nm on the ordinate. Data are presented in (a) and (f) using goat 4g2 serum; in (b) serum from a CAEV-negative goat, 2g1; in (c) serum from a CAEV-positive goat, 19g1; in (d) a control goat anti-gp120 serum; and in (e) a control mouse anti-gp120 monoclonal antibody.

To this end, we undertook a series of adsorption experiments in which we coated plastic with the gp135 protein antigen, added goat serum to remove reactive immunoglobulins, and then examined the depleted sera by ELISA assay, to determine which antibody specificities had been removed or retained. Since the gp135-4g2 protein has a 6⫻ His-tag carboxyl-terminus and since we found several goat sera had high nonspecific backgrounds in ELISA assays, we always preadsorbed goat sera with a 6⫻His-tag control protein (Ross River Virus E2) to remove His-tag-reactive antibodies. We found that no gp135- or gp120-specific antibodies were removed by this procedure (data not shown). This serum, designated 4g2 (RR), was then adsorbed using gp135-4g2 protein coated on plastic. The depleted serum was tested for the presence of antibodies which can interact with gp135-4g2 and gp120 IIIB-r using ELISA assays. Fig. 3a shows clearly that the adsorption step

removed a small, but significant, amount of gp135-4g2 reactivity, and most importantly, all the gp120 specificities (Fig. 3b). We, therefore, conclude that the gp135-specific antibodies from the 4g2 goat do indeed cross-react with gp120. Since we have clearly shown that the gp135-4g2 protein interacts with gp135-specific antibodies having gp120 reactivities, we reasoned that the reverse should be true—i.e., that adsorption with the gp120 protein should deplete 4g2 serum of gp135-specific antibodies. The results of the ELISA assays are shown in Figs. 3c and d. These ELISAs were performed at greater dilutions of antiserum than in Figs. 3a and b since the gp120-reactive antibodies constitute a minor fraction of the total gp135specific antibodies. The data in Fig. 3c demonstrate that the gp120 protein interacts and therefore depletes gp135 specificities, as would be predicted of cross-reactive an-

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Fig. 3. Antibodies specific for gp135 cross-react with gp120. The antigens used in each ELISA assay are identified above each panel and the antigens used for adsorption are identified in the right-hand box: ■ denotes control 4g2 (RR) serum before adsorption; Œ denotes after adsorption with gp135-4g2 protein; F denotes adsorption with gp120 IIIB-r protein. Serum dilution of 4g2(RR) is shown on the abscissa and the absorbance at 405 nm is shown on the ordinate.

tibodies, while all the gp120 reactivity is removed as would be expected (Fig. 3d). In an alternate approach, we undertook inhibition ELISA experiments in which we incubated soluble antigen (gp1354g2) with 4g2 serum to inhibit the binding of 4g2 antibodies to plastic-bound antigens (gp135-4g2 or gp120 IIIB-r). If gp135-4g2 antibodies are capable of cross-reacting with gp120 IIIB-r, then incubating 4g2 serum with increasing amounts of soluble gp135-4g2 should progressively remove gp120-reactive antibodies. The results are presented in Fig. 4. Since the gp120-reactive antibodies comprise a minor fraction of the total gp135-specific antibodies, different concentrations of inhibiting antigen are used in Figs. 4a and b. From these data, soluble antigen is clearly inhibitory, demonstrating unequivocally that the gp135-specific antibodies cross-react with gp120, thereby corroborating our conclusions from the results outlined in Fig. 3. Having shown that the gp135-4g2 antibodies do indeed cross-react with gp120, we wished to further characterize these immunoglobulins— can they interact with gp120 IIIB expressed in mammalian cells if the carbohydrate is removed? Goat gp120-reactive antibodies can interact with deglycosylated gp120 IIIB-m Our experiments have established that gp135-specific antibodies can react with gp120 protein from several viral

strains, provided it is expressed in insect cells, but not with gp120 protein synthesized in mammalian cells. We conclude, therefore, that the state of glycosylation of the protein is critical to this antibody reactivity. Proteins expressed in insect cells contain high mannose or truncated trimannosyl N-linked glycans (Jenkins et al., 1996; Kuroda et al., 1990).

Fig. 4. Antibodies specific for gp135 cross-react with gp120. The antigens used in these inhibition ELISA assays are indicated above each panel. The concentration of gp135-4g2 added to the goat 4g2 serum is denoted on the abscissa and the OD at 405 nm is denoted on the ordinate.

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Fig. 5. Goat gp120-reactive antibodies can interact with linear amino acid epitopes. Chemiluminescent Western blots using 4g2 serum with the following antigens: lanes 1 and 2, gp135-63; lanes 3 and 4, gp135-4g2; and lanes 5 and 6, gp120 IIIB-m. Proteins in lanes 2, 4, and 6 were deglycosylated using peptide-N-glycosidase F. Molecular weights are indicated on the left-hand side.

In contrast, proteins from mammalian cells can be more heavily glycosylated because they contain complex N-linked carbohydrate side chains (Davis et al., 1993; Kornfeld and Kornfeld, 1985). Since our antibodies do not recognize the SIV envelope protein isolated from insect cells, we hypothesized that their reactivity is not specific for insect carbohydrate, per se, but rather is at least partially dependent on the protein sequence of gp120 which is more exposed in the baculovirus-derived product. To examine the role of carbohydrate in antibody activity, we undertook a chemiluminescent Western blot analysis of the 4g2 goat serum using gp120 IIIB-m in its glycosylated and deglycosylated forms. The results are shown in Fig. 5. 4g2 antibodies react with both the glycosylated and the PNGase-treated forms of gp135-63 (lanes 1 and 2) and gp135-4g2 (lanes 3 and 4) and with deglycosylated (lane 6), but not the native form (lane 5), of gp120 IIIB-m protein. These results substantiate the ELISA data in Fig. 2 and indicate that at least some of the goat gp120-reactive antibodies react with the protein portion rather than carbohydrate moieties of gp120. In addition, since the analysis was done under reducing conditions, a population of the 4g2 antibodies must interact with linear epitopes on gp120.

Discussion In this article, we show that some goats infected with CAEV can generate gp135-specific antibodies which crossreact with baculovirus-derived gp120 protein from several strains of the HIV virus. This is an unexpected and intriguing finding. At an initial glance, gp135 and gp120 proteins appear to be disparate molecules, differing in molecular weight and lacking primary sequence homology (Crawford et al., 1980; Modrow et al., 1987; Valas et al., 2000). Nevertheless, both are unusually heavily glycosylated, both are noncovalently bound to their respective transmembrane domains, and both mediate viral entry into target cells. Indeed, they share two short sequence motifs, suggesting structural similarities (Hotzel and Cheevers, 2000, 2001). Thus, their apparent unrelatedness may not necessarily indicate independent ter-

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tiary structures, since proteins with no primary sequence homology can fold into very analogous 3D structures or domains (Branden and Tooze, 1999; Pancino et al., 1994). Gp135 and gp120 therefore may share structural elements which create common conformational or linear epitopes that generate this surprising cross-reaction. At present, we cannot formally distinguish between cross-reaction and molecular mimicry as the basic explanation for gp135-specific antibody recognition of gp120. Experiments are ongoing to clarify this and to define the epitopes at a molecular level. We have demonstrated that the observed cross-reactivity of the 4g2-derived antibodies is carbohydrate-dependent, since the goat gp120-reactive antibodies interact with gp120 protein expressed in insect cells or with deglycosylated gp120, derived from mammalian cells. These data imply the cross-reactive antibodies recognize epitopes which reside in heavily glycosylated regions of gp120 and that removal of carbohydrate unmasks epitopes, at least some of which depend on the the primary sequence of gp120. Glycosylation plays a crucial role in enabling the gp120 monomer to evade an effective immune response (Johnson and Desrosiers, 2002; Poignard et al., 2001). The carbohydrate moieties, per se, have low immunogenicity and are so numerous on gp120 that they conceal receptor-binding sites from interaction with potentially neutralizing antibodies (Wei et al., 2003). Since these critical sites are only transiently exposed when gp120 undergoes a conformational change upon binding to its CD4 receptor (Kwong et al., 1998; Rizzuto et al., 1998; Sullivan et al., 1998; Wyatt et al., 1998; Wyatt and Sodroski, 1998), removal of carbohydrate from gp120, to increase protein epitope accessibility, has been proposed as a vaccine strategy (Lee et al., 1992; Reitter et al., 1998). Thus, the fact that the 4g2 antibodies cannot react with gp120-m does not necessarily detract from their potential utility. In summary, we have demonstrated cross-reactivity between gp120 and another lentiviral envelope protein. We are presently determining whether the gp120-reactive antibodies can, in fact, neutralize the HIV virus and are identifying the epitopes on gp120 with which they interact.

Materials and methods Goat sera and virus Sera were prepared from the blood of 46 goats at 20 ranches in southern and central California. Peripheral mononuclear cells from goat 4g2 were cocultured with goat synovial membrane (GSM) cells. The virus was amplified for 14 days and isolated from supernatant by ultracentrifugation. Viral RNA was prepared using TRIzol LS (Invitrogen, Carlsbad, CA). cDNA was generated and amplified by RACE technology and the envelope gene was cloned using a TOPO XL PCR cloning kit (Invitrogen).

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Antigens The following antigens were used in ELISA and Western blot assays: HIV gp120 IIIB envelope protein produced in mammalian cells (gp120 IIIB-m) (Advanced Biotechnologies, Inc., Columbia, MD; ABI); HIV gp120 IIIB protein produced in baculovirus-infected insect cells (gp120 IIIB-r) (Trinity Biotech, Inc. Carlsbad, CA); simian immunodeficiency virus envelope protein produced in insect cells (Trinity Biotech), HIV gp120 MN protein produced in insect cells (gp120 MN-r) (ABI); HIV gp120 CM protein produced in insect cells (gp120 CM-r); HIV gp120 SF2-m protein produced in mammalian cells, both obtained from the NIH AIDS Research and Reference Reagent Program, and ovalbumin protein (Sigma, St. Louis, MO). The envelope protein of the CAEV-63 virus, gp135-63, was purified by affinity chromatography from the culture medium of GSM cells (Kemp et al., 2000). The envelope protein of the 4g2 virus, gp135-4g2 (amino acids 1–549), with a 6⫻ His-tag at the carboxyl-terminus was expressed in a lytic baculovirus/insect cell expression system and purified using Ni-NTA chromatography as previously described (Lebron et al., 1998). The protein was analyzed by SDS–PAGE and shown to be 95% pure. The Ross River virus E2 protein, having a 6⫻His-tag carboxyl-terminus, was used as a control protein. ELISA The reactivities of goat sera to gp135-63, gp135-4g2, gp120 IIIB-r, gp120 IIIB-m, gp120 MN-r, gp120 CM-r, gp120 SF2-m, SIV, and OVA were determined by indirect enzyme-linked immunosorbent assays (Hornbeck, 2001). A mouse anti-gp120 monoclonal antibody which recognizes the V3 loop of HIV gp120 (Trinity Biotech) and a goat anti-gp120 antibody (NIH AIDS Research and Reference Reagent Program, Cat. No. 364) were used as controls. All incubations were done at room temperature. Immulon 4 ELISA plates (Dynatech, Chantilly, VA) were coated overnight with 50 ␮l/well of antigen in PBS/0.05% sodium azide at a concentration of 2 ␮g/ml. The alkaline phosphatase conjugated detection antibody (rabbit anti-mouse H⫹L; Pierce, Rockford, IL) or rabbit anti-goat H⫹L (Pierce) was used at a dilution of 1:10,000. P-nitrophenyl phosphate (PNPP) substrate (Pierce) was used according to the manufacturer’s instructions at 100 ␮l/well. Plates were developed for 1 h and read at 405 nm on an Emax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA). For adsorption ELISA experiments, goat sera were adsorbed on plastic coated with antigen, to remove specific antibodies. Immulon 4 ELISA plates (Dynatech) were coated with antigen, washed, and blocked, as described above. Serum to be adsorbed was diluted in ELISA blocking buffer (Hornbeck, 2001) 1:100, 1:500, 1:1000, added to the antigen-coated ELISA plate (70 ␮l/well), and incubated at room temperature, with shaking. Serum was adsorbed for

6 h, being transferred to a new set of antigen-coated wells every 2 h. The serum was then removed and stored at ⫺20°C. It was then analyzed by ELISA according to the protocol described above. In the inhibition ELISA assays, gp135-4g2 protein was used to block the binding of 4g2 antibodies to plastic-bound antigen. Varying concentrations of gp135-4g2 protein and an equal volume of a 1:25 dilution of serum were incubated at room temperature for 1 h with rocking. Dilutions of both protein and serum were made using ELISA blocking buffer (Hornbeck, 2001). This serum was then tested directly for reactivity to gp135-4g2 and gp120-IIIB-r in ELISA assays, as described above. PNGase digestion Peptide-N-glycosidase F (PNGase; GlycoPro, San Leandro, CA) was used to remove N-linked high-mannose oligosaccharides from gp135 and gp120 IIIB-m. Digestion was carried out for 16 h as described (Ozyoruk et al., 2001). Western blot Serum from goat 4g2 was assayed for reactivity to gp120-IIIB-m, gp135-63, and gp135-4g2 with and without PNGase treatment by Western blot under reducing conditions. Antigen was diluted 1:2 in 2⫻ sample buffer (0.06 M Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromphenol blue, 5% ␤-mercaptoethanol) and heated at 100°C for 5 min. Samples were electrophoresed at 200 V for 35 min in 10% polyacrylamide gels (Tris–HCl Ready Gel, Bio-Rad, Hercules, CA) using a Mini-Protean 3 Cell (Bio-Rad) and were then transferred to nitrocellulose membranes in 1 h at 100 V, 350 mA. All subsequent incubations and washings were done with constant rocking. Membranes were washed for 30 min at room temperature with TBS (20 mM Tris, 0.1 M NaCl, pH 7.4)/0.3% Tween 20 (Sigma) and then blocked for 30 min with TBS/3% BSA/0.05% Tween 20. Serum was diluted 1:200 in TBS/3% BSA/0.05% Tween 20, added to the membrane, and incubated 1 h at room temperature. The membrane was then washed four times in TCBS, pH 5.5 (20 mM sodium citrate, 500 mM NaCl, 0.05% Tween 20), incubating each wash for 5 min at room temperature. Horseradish peroxidase (HRP) conjugated Protein G (Bio-Rad) was used for detection. Membranes were incubated at room temperature for 90 min with HRP-conjugated Protein G diluted 1:500,000 in TCBS containing 1% gelatin. Membranes were then washed three times with TBS/0.05% Tween 20/1% gelatin in a 55°C water bath, followed by three washes (5 min each) in TBS/0.05% Tween 20 at room temperature. Immunoblots were developed using the chemiluminescent substrate system, SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). Blots were incubated for 5 min at room temperature in the working solution provided by the kit, before being imaged on a Fluor-S Max Imager (Bio-Rad).

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Acknowledgments We thank Dr. John Zaia (City of Hope National Medical Center), Drs. Gunther Dennert, Alexandra Levine, Marek Nowicki, Robyn McGuire (USC), and Dr. Pamela Bjo¨ rkman (Caltech) for helpful discussions, and Linda Enger for goat handling and venipuncture. This work was supported by NIH Grant R21AI45396, by the Campbell Foundation, Ft. Lauderdale, FL (to A.D.), and by Surro Immunology, Inc., Encino, CA. We acknowledge the NIH AIDS Research and Reference Reagent Program for providing several reagents which greatly accelerated our progress.

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